Schistosoma mansoni miracidia transformed by particle bombardment infect Biomphalaria glabrata snails and develop into transgenic sporocysts

Experimental Parasitology 105 (2003) 174–178 www.elsevier.com/locate/yexpr

Research Brief

Schistosoma mansoni miracidia transformed by particle bombardment infect Biomphalaria glabrata snails and develop into transgenic sporocysts Oliver Heyers,a Anna K. Walduck,b Paul J. Brindley,c Wilfrid Bleiß,a Richard Lucius,a Tomislav Dorbic,d Burghardt Wittig,e and Bernd H. Kalinnaa,* a

e

Department of Molecular Parasitology, Institute for Biology, Humboldt University Berlin, 10115 Berlin, Germany b Max Planck Institute for Infection Biology, 10117 Berlin, Germany c Department of Tropical Medicine, Tulane University, Health Sciences Center, New Orleans, LA 70112, USA d Centrum Somatische Gentherapie an der Freien Universit€at Berlin, Fachbereich Humanmedizin, UKBF and Geniotronic AG, Berlin, Germany Centrum Somatische Gentherapie an der Freien Universit€at Berlin, Fachbereich Humanmedizin, UKBF and Mologen Holding AG, Berlin, Germany Received 5 June 2003; received in revised form 8 September 2003; accepted 4 November 2003

Abstract Miracidia (and adults) of Schistosoma mansoni which had been subjected to particle bombardment with a plasmid DNA encoding enhanced green fluorescent protein (EGFP) under control of the S. mansoni heat shock protein 70 (HSP70) promoter and termination elements were shown to express the reporter gene. Bombarded miracidia were able to penetrate and establish in Biomphalaria glabrata the intermediate host snail. Gold particles could be detected in the germ balls of parasites in paraffin-sections of snail tissue. The bombarded miracidia were able to develop normally and to transform into mother sporocysts. Reporter gene activity could be determined at 10 days post-infection by RT-PCR in snail tissues, but not by microscopy or Western blot which probably reflected sub-optimal expression levels of constructs. Our findings indicated that it is feasible to return transgenic miracidia to the life cycle, a crucial step for the establishment of a transgenesis system for schistosomes. Ó 2003 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: schistosome, miracidium, sporocyst, particle bombardment, gold particles, transgene, Biomphalaria glabrata; germ ball; EGFP, enhanced green fluorescent protein; RT-PCR, reverse transcription-polymerase chain reaction; gDNA, genomic DNA; PBS, phosphate buffered saline; TBS, Tris buffered saline; FCS, fetal calf serum; DEAE, diethylaminoethyldextran

Schistosomes are considered the most important of the human helminths in terms of morbidity and mortality (Engels et al., 2002). A tractable transfection system would represent a significant advance in our ability to develop new interventions for control of schistosomiasis (Boyle and Yoshino, 2003; Davis et al., 1999). Schistosoma japonicum and Schistosoma mansoni sporocysts can be generated in vitro by transforming miracidia (reviewed in Coustau and Yoshino, 2000), and cultured sporocysts have been transiently transfected with pBluescriptbased plasmids expressing green fluorescent protein (GFP) (Wippersteg et al., 2002a,b), demonstrating that it is possible in principle to generate transgenic parasites. However, it is not clear yet whether a transgenic line of schistosomes could be established from these transiently transfected sporocysts. At least two important obstacles on the path to the development of a reliable, tractable transgenesis system for schistosomes now need to be surmounted: (1) development of appropriate constructs to achieve stable integration into the schistosome

* Corresponding author. Fax: +49-30-2093 6051. E-mail address: [email protected] (B.H. Kalinna).

0014-4894/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2003.11.001

genome and (2) a demonstration that the life cycle can be completed by genetically modified parasites. The life cycle of the schistosome includes two free-living, self-reliant, and mobile infective stages. These are the miracidium, which seeks out and directly infects the intermediate host snail, and the cercaria, which accomplishes infection of the mammalian, definitive host by direct penetration of host skin when a definitive host comes into contact with water contaminated with cercariae. These are the only two stages that could be reintroduced into the life cycle using a natural route of infection, thereby obviating the inefficient and laborious tasks of surgically implanting transformed sporocysts back into snails and/ or surgically implanting adult schistosomes into the portal system blood vessels of mice (e.g., Cheever et al., 1994; Jourdane et al., 1985). Here we subjected miracidia of S. mansoni to particle bombardment and have been able to show that miracidia bombarded with DNAcoated gold particles can directly and naturally infect the intermediate host snail Biomphalaria glabrata and establish as sporocysts in a natural fashion. Further we were able to demonstrate transgene transcription of enhanced GFP in both adult worms and snails infected with transformed sporocysts.

O. Heyers et al. / Experimental Parasitology 105 (2003) 174–178 Schistosoma mansoni (Puerto Rican strain) was propagated in the laboratory as described (Copeland et al., 2003). Snails were maintained at 26 °C in aerated water and fed with lettuce leaves, in a room with a light/darkness cycle of 12 h each. At 8 weeks after S. mansoni infection, mice were killed, adult worms were perfused from the mesenteric veins with PBS, and the livers were removed. Adult worms were washed three times in RPMI 1640 and maintained in vitro in RPMI 1640 + 10% FCS supplemented with penicillin/streptomycin at 37 °C in 5% CO2 . The infected livers were forced through a plastic mesh to release the schistosome eggs, after which the eggs were washed in cold sterile 1.8% NaCl to remove host tissues and debris. The 1.8% NaCl was replaced with sterile water to induce the eggs to hatch, miracidia were collected from the water, and concentrated by centrifugation at 500 rpm. For particle bombardment (biolistics), approximately 500 miracidia or 20 male, adult worms were evenly spread onto a polycarbonate membrane (Transwell, Costar), water or medium was removed and target schistosomes on the polycarbonate membranes were positioned in a Biolistic PDS-1000/HE Particle Delivery System (BioRad Laboratories GmbH, M€ unchen, Germany). Biolistic parameters were 15 in. Hg of chamber vacuum, target distance of 3 cm (stage 1), 900 psi (miracidia) or 1800 psi (adult worms) particle acceleration pressure, and 1.0 lm gold microcarriers (Bio-Rad). Gold microcarriers were prepared, and circular plasmid DNA was precipitated onto the gold using methods recommended by Bio-Rad with the following modification; 0.6 mg of gold particles carrying
5 lg of plasmid DNA was used per bombardment. To engineer plasmid constructs for these transient transfection studies, a 532 bp fragment comprising the promoter region of the gene encoding the 70 kDa heat shock protein (HSP70) of S. mansoni (GenBank Accession No. L02415) (Neumann et al., 1992) was amplified from S. mansoni genomic DNA using a 50 primer, 50 -GCGC GGAATTCGATTAGCACTAAG-30 , and a BamHI-linked 30 primer, 50 -GCGCGGGATTCTCCAAGATATGTTAAGAC-30 . Genomic DNA (gDNA) was isolated from adult schistosomes and from snails as described (Copeland et al., 2003). The HSP70 30 end terminator region was isolated by amplifying a 760-bp fragment from gDNA with the XbaI-linked forward-primer, 50 -CCCCCTCTAGACCTATAATTGT TGTGATAAATTG-30 , and a HindIII-linked reverse-primer, 50 -CC CCCAAGCTTAGAACCCTTCCACGGTGTG-30 . To isolate an EGFP-encoding fragment, pEGFP-N1 (Clontech) was digested with BamHI and XbaI. The 720-bp fragment was purified by agarose electrophoresis. The three different fragments were ligated into pUC19 digested with the appropriate restriction enzymes. A map of this construct is presented in Fig. 1A. The construct was tested in COS7 cells and found to be functional in that
5% of the COS7 cells ex-

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hibited EGFP fluorescence (not shown). To generate a control plasmid, pEGFP-N1 (Clontech) was modified by deletion of the CMV promoter. Total RNA was extracted from snails or adult S. mansoni worms using the RNeasy kit (Qiagen, Hilden, Germany). DNAsetreated RNA was used as a template for oligo(dT) primed reverse transcription. The resulting cDNA was used as the template in PCR experiments using primers specific for the EGFP, 50 -ATGG TGAGCAAGGGCGAG-30 and 50 -TTACTTGTACAGCTCGTC-30 , expected target size, 740 nt. One microliter of the first PCR was used as template for a second round of nested PCR using primers 50 -GC AGTGCTTCAGCCGCTA-30 and 50 -TAGTGGTTGTCGGGCAG C-30 , expected target size, 396 nt. Negative control reactions were carried out using: (a) the non-transcribed DNAse-treated RNA and (b) gDNA of uninfected snails as templates. DNA of plasmid pEGFP-N1 served as the positive control. Adult worms were bombarded twice within a few minutes, culture medium (above) was added, and the worms maintained in vitro for 24 h. Adult schistosomes were then heat shocked at 42 °C for 4 h and cultured for an additional 24 h at 37 °C before examination for EGFP. Larval (miracidia, sporocysts) schistosomes were not heat shocked because the schistosome HSP70 promoter is constitutively active in these developmental stages (Neumann et al., 1993). Fluorescence microscopy of bombarded worms was performed using a Zeiss Axioplan fluorescence microscope (Oberkochen, Germany) with a blue excitation filter set (No. 487909) and/or with a Leica TCS NT laser scanning, confocal microscope, as described (Wippersteg et al., 2002a,b). Small regions of EGFP fluorescence which co-localized with gold particles could be distinguished from the relatively high background fluorescence only by using the laser scanning microscope. By contrast, green (EGFP) fluorescence was not observed in untreated control worms, worms that were bombarded with unlabelled gold, or with gold coated with the promoter-less plasmid. Here only background fluorescence was visible. EGFP fluorescence was not detectable by normal fluorescence microscopy in any experiment (i.e., by using a Zeiss Axioplan fluorescence microscope); it was detected only with the confocal microscope. This inability to detect fluorescence with normal fluorescence microscopy has also been noted by others (Wippersteg, personal communication), and may reflect poor transcription/translation and/or incorrect folding of the EGFP. Transfection rates varied between 0.5 and 1.0% in independent experiments, although prominent fluorescence could only be detected in
0.1% of bombarded organisms. The fluorescence pattern observed had no obvious specific distribution but tended to be more often seen at the oral sucker (Fig. 1B) or at the posterior end of the worm, in agreement with Wippersteg et al. (2002a). In these cases, it was relatively straightforward to distinguish

Fig. 1. (A) Schematic representation of the HSP70-GFP-HSP70 plasmid vector used for transfection. The vector has a pUC19 plasmid backbone. The insert contains the promoter (50 HSP70), EGFP gene and terminator (HSP70 30 ). Not drawn to scale. (B) Confocal microscope image of a male worm showing EGFP expression in the vicinity of the oral sucker 72 h after bombardment. For this bombardment, 1.0 lm gold microcarriers and a helium pressure of 1800 psi were used. The inset presents a scanning electron micrograph of the anterior of an adult S. mansoni male, with the boxed region indicating the location shown in the fluorescence micrograph. A scale bar indicates the size. (C) EGFP mRNA expression in adult worms after particle bombardment. Lanes 1, HSP70-promoter plasmid; 2, Marker; 3, promoter-less plasmid control. The arrow indicates a 740 bp band corresponding to the expected size of the EGFP RT-PCR product.

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the EGFP fluorescence from background signals. To verify the expression of EGFP on the transcriptional level, an EGFP-specific RTPCR with RNA extracted from EGFP-expressing worms produced amplicons of the expected size, 740 bp, confirming the transcription of the EGFP in bombarded worms (Fig. 1C). Infection of snails of
5 mm diameter shell size with transformed miracidia was accomplished by incubation of the snails with approximately 150–250 transformed miracidia in 0.5 ml of water for 3 h under bright light. This high infection rate regimen was employed because we anticipated that several only of the transfected sporocysts would develop in each snail and because we did not know what percentage of the bombarded miracidia would be capable of infection. Invasion of snails by bombarded miracidia was monitored by microscopic observation. The snails survived this infection regimen, after which they were maintained for several weeks. In some instances, bombarded miracidia were cultured overnight in medium MEMSE-J + 10% fetal calf serum (FCS) for transformation in vitro into primary (mother) sporocysts (Bayne et al., 1994; Jourdane et al., 1985). To determine if miracidia could withstand particle bombardment, miracidia were bombarded with unlabelled gold microcarriers at 900 psi. Subsequently, they were washed, transferred into MEMSEJ + 10% FCS, and cultured at 26 °C in air. About half the bombarded miracidia survived, as adjudged by light microscopy for normal larval movements (not shown). These miracidia transformed into mother sporocysts within 24 h. Single gold particles or gold aggregates were readily observed in
15% of these mother sporocysts. Further, microscopic examination of these sporocysts revealed the gold particles in different focal planes (Figs. 2A–C), indicating that the microcarriers

had penetrated into deeper tissue layers. Mother sporocysts containing gold particles appeared to be otherwise intact and were indistinguishable in other ways from control, in vitro derived mother sporocysts (not shown). Indeed, these sporocysts containing gold particles were metabolically active as judged by the movement of the flame cells, peristaltic movements, and depletion of culture media during the course of the experiment. These findings indicated that many miracidia survive the biolistics procedure and, second, that bombarded miracidia can transform into mother sporocysts in vitro. To examine whether transgenic parasites could survive in the intermediate host, miracidia which had been bombarded with gold particles were used to infect B. glabrata snails. Snails were exposed to bombarded miracidia and the progression and penetration of the bombarded miracidia was monitored by light microscopy (Zeiss Axioplan microscope). Most miracidia had penetrated the snails by 3 h. Histological sections of the infected snails and of uninfected control snails were prepared at 10–14 days after infection. Shells were removed from the snails using forceps, the snails fixed in BouinÕs fluid, washed in 70% ethanol, dehydrated in 100% ethanol, cleared with xylol, and embedded in paraffin (Paraplast Plus, Sherwood Medical, St. Louis, MO) (Lemos and Andrade, 2001; Romeis, 1989). Five micrometer snail tissue sections were cut and then stained with haematoxylin and eosin. Mother sporocysts could be observed in the tissues of the snails (Fig. 3) and their structure and appearance were similar to that described for normal sporocysts by Lemos and Andrade (2001). Germ balls were clearly visible and gold particles (optically dense, 1–2 lm diameter) were located in the sporocyst tissues (Figs. 3A–C). The nuclei of the germ ball cells were large and euchromatic and hetero-

Fig. 2. Transmission micrograph of in vitro generated mother sporocyst that has developed from a miracidium at 72 h after the miracidium was subjected to particle bombardment with gold particles (not coated with DNA). Arrows indicate the position of gold particles (particles are 1.0 lm diameter). The same sporocyst is shown in the three panels in different focal planes. Scale bars indicate sizes.

Fig. 3. Haematoxylin and eosin-stained paraffin section of B. glabrata snail 2 weeks after infection with miracidia transformed by particle bombardment with gold micro-carriers. (A) Overview section through the mouth, mid-body, head–foot, and hepatopancreas/kidney region of the snail. The box outlines the kidney region that is infected with sporocyst. This boxed region is shown at higher magnifications in (B and C). The locations of other key anatomical features are indicated by text. (B) Higher magnification of snail tissues shown in (A); the regions in and around the box outline include developing mother sporocysts. The radula of the snail is prominent in the upper left of the panel. The locations of other key anatomical features are indicated by text. (C) Higher power magnification of snail tissues from (B), with a close-up image of sporocysts. The arrows show the position of gold particles in close proximity to the germ ball cells in the sporocysts. Scale bars are included at the top of each panel.

O. Heyers et al. / Experimental Parasitology 105 (2003) 174–178 chromatin was apparent at the nuclear membrane (Figs. 3B and C, indicative of elevated metabolic activity in these cells. The prominent nucleoli were also enlarged and showed lighter areas (Fig. 3C), indicative of transcriptional activity (Smetana, 1974; Underwood, 1990). In many cases, the gold particles co-localized with the germ ball cells (Fig. 3C). This co-localization and the obvious integrity of the sporocysts demonstrated that it is possible to transform miracidia by particle bombardment and to use miracidia transformed in this way to infect B. glabrata. Thus, these techniques represent useful tools for the establishment of daughter lines of transformed schistosomes. Next, we examined snails infected with miracidia that had been bombarded with gold particles coated with plasmid constructs encoding the EGFP reporter under control of the schistosomal HSP70 gene regulatory regions, or with a control promoter-less plasmid. We failed to observe EGFP in tissues of these snails by normal or confocal fluorescence microscopy. Neither were we able to detect EGFP protein by immunoblot analysis of snail tissues employing an anti-EGFP antibody (not shown). However, when we examined the snail tissues for transcription of mRNA encoding EGFP using an EGFP-specific nested RT-PCR with total RNA from infected snails, we detected amplicons of the expected size, 396 bp (Fig. 4). This confirmed that the EGFP transgene introduced into miracidia by particle bombardment was indeed transcribed in sporocysts developing in these snails. To summarize, here we have demonstrated that miracidia of B. glabrata can withstand and survive particle bombardment with gold microcarriers carrying plasmid transgenes and that mother sporocysts of S. mansoni were vital and developed in vitro from miracidia that survived particle bombardment. In addition, we showed that gold particles had entered into the miracidia at different depths. Indeed, because the detection of single gold particles is difficult, we speculate that the actual transformation efficiency may have been higher than we observed. Also, we have demonstrated that bombarded miracidia were able to infect B. glabrata snails. Numerous developing sporocysts were evident within organs and tissues of the snails by about 2 weeks after infection, and their morphology was indistinguishable from that described by Lemos and Andrade (2001) for natural (non-transformed) S. mansoni sporocysts in B. glabrata. Thus, we assume that sporocysts which developed from bombarded miracidia were completely vital. Also, many gold particles were visible and co-localized with germ ball cells in these sporocysts. Since it has also been shown that organelles

Fig. 4. EGFP mRNA expression in tissues from a snail infected with miracidia transformed by particle bombardment by gold microcarriers coated with the S. mansoni HSP70 promoter-EGFP plasmid. Lanes 1 and 3: nested PCR with EGFP-specific primers; lanes 2 and 4: negative control; no reverse transcription prior to PCR; lane 5: negative control; nested PCR with EGFP-specific primers using genomic DNA from an uninfected snail as template; lane 6: positive control PCR with plasmid DNA; lane 7: Marker. Lanes 1, 2 and 3, 4 correspond to two independent experiments.

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inside cells can be transfected by biolistic methods (Butow et al., 1996; Daniell, 1993; Lorito et al., 1993; Rohou et al., 2001; Ruf et al., 1997), it seems feasible that transfection of the sporocyst germ balls can be achieved. Transfection of the totipotent cells that constitute the germ balls of sporocysts will, theoretically, lead to the formation of transgenic daughter sporocysts. This, in turn, should facilitate the development of a transgenic line of schistosomes. The use of adult worms for transfection of schistosomes presents an inherent problem in that adult worms cannot easily be introduced back into the life cycle. Adult worms would have to be implanted into the mesenteric veins of mice, which is technically difficult. Adult worms are, however, useful for testing schistosomal expression plasmids. Moreover, the use of mother sporocysts for transformation experiments as described by Wippersteg et al. (2002a,b) may be less than optimal. Although sporocysts can be generated in vitro from eggs and methods for their long term culture and co-culture with a B. glabrata embryo (bge) cells line have been developed (Bayne et al., 1994; Ivanchenko et al., 1999; Jourdane et al., 1985), implantation of in vitro cultured sporocysts into the snails is difficult and survival rate of snails and implanted sporocysts is low. The coding region of the EGFP reporter used here contains mutations which improve the intensity of green fluorescence. Furthermore it contains other mutations which optimize protein folding and solubility at 37 °C, apparently making it suitable for expression in adult schistosomes, which are cultured at this temperature. Although EGFP has been reported to have a 35-fold higher intensity than GFP (Heim and Tsien, 1996), we were only able to detect EGFP fluorescence using laser scanning microscopy. The expression pattern we observed was comparable to that reported by Wippersteg et al. (2002a). After heat shock, EGFP signals in adult worm tissues were seen preferentially in tegumental areas, areas around the head sucker and at the posterior end, and co-localized with gold particles in most cases. Expression of EGFP in schistosomes is therefore possible but does not constitute a significant improvement over the previous reports. Possibly the weak EGFP signal results from low expression and/or incorrect folding of EGFP in schistosomes. It is known, that the removal of a cryptic intron in the GFP coding sequence is required to achieve prominent fluorescence in transgenic Arabidopsis thaliana (Haseloff et al., 1997). Introducing introns into the reporter sequence enhances GFP expression in Caenorhabditis elegans (Fire et al., 1990). Similar modifications to constructs might enhance EGFP performance in schistosomes. In infected snail tissues, EGFP could not be detected on the protein level by immunoblot analysis, likely because the amount of EGFP translated was below detectable levels in preparations of the extracts of total snail proteins. On the other hand, EGFP transcripts in the snails could be detected by nested RT-PCR, indicating that the reporter gene was transcribed in these snails. Strong promoters that are constitutively expressed in all schistosome developmental stages have not yet been characterized and this remains as a serious impediment for development of a tractable transgenesis system. The promoters used here and by Wippersteg et al. (2002a,b) are developmentally regulated, and may need specific, but as yet undefined, conditions for activity. This could be an additional explanation for the poor expression of the EGFP (or GFP) and the inability to detect fluorescence using conventional fluorescence microscopy, which is straightforward in other systems involving, for example, Drosophila melanogaster or C. elegans. For future studies with transgenesis in schistosomes, the deployment of additional reporters or promoters may improve expression levels of the transgenes. Because numerous genomic sequences of schistosomes are now becoming available, we can anticipate the introduction in the near future of more appropriate endogenous gene promoters. Finally, the findings reported here present proof of the principle that miracidia can be transformed by biolistics procedures using gold microparticles coated with transgene constructs, and that the transformed miracidia retain the capacity to infect snails and to begin the propagation of transgenic schistosomes. Yet simple plasmids located episomally in the germ ball cell cytoplasm of transfected sporocysts

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will not likely or reliably lead to chromosomal integration of reporter transgenes. In order to enhance the prospects for chromosomal integration of schistosome transgenes, we are currently investigating the utility of mobile genetic elements including transposons, retrotransposons, and pseudotyped retroviruses as transfection vectors. RNA interference techniques in schistosomes (e.g., Skelly et al., 2003) may also be compatible with biolistics procedures for transgenesis of schistosomes.

Acknowledgments We thank Grit Meusel for laboratory assistance, Anorte Marko for preparing histological specimens and Ute Mackenstedt for interpretation of snail histology. This study was supported by a grant from the Deutsche Forschungsgemeinschaft (KA 866/2-1) to B.H.K. P.J.B. is a recipient of a Burroughs Wellcome Fund scholar award in Molecular Parasitology and is supported by an Infrastructure Grant from the Ellison Medical Foundation (Award No. ID-IA-0037-02).

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